46 Modern Food Microbiology Table 3–4 Relationship between Water Activity and Concentration of Salt Solutions Sodium Chloride Concentration Water Activity Molal Percent, w/v 0.995 0.99 0.98 0.96 0.94 0.92 0.90 0.88 0.86 0.15 0.30 0.61 1.20 1.77 2.31 2.83 3.33 3.81 0.9 1.7 3.5 10 13 16 19 22 Source: From The Science of Meat and Meat Products, by the American Meat Institute Foundation W.H Freeman and Company, San Francisco; copyright c 1960 Table 3–5 Approximate Minimum a w Values for Growth of Microorganisms Important in Foods Organisms aw Groups Most spoilage bacteria Most spoilage yeasts Most spoilage molds 0.9 0.88 0.80 Specific Organisms Clostridium botulinum, type E Pseudomonas spp Acinetobacter spp Escherichia coli Enterobacter aerogenes Bacillus subtilis Clostridium botulinum, types A and B Candida utilis Vibrio parahaemolyticus Botrytis cinerea Rhizopus stolonifer Mucor spinosus 0.97 0.97 0.96 0.96 0.95 0.95 0.94 0.94 0.94 0.93 0.93 0.93 ∗ Perfect Organisms Groups Halophilic bacteria Xerophilic molds Osmophilic yeasts Specific Organisms Candida scottii Trichosporon pullulans Candida zeylanoides Geotrichum candidum Trichothecium spp Byssochlamys nivea Staphylococcus aureus Alternaria citri Penicillium patulum Eurotium repens Aspergillus glaucus∗ Aspergillus conicus Aspergillus echinulatus Zygosaccharomyces rouxii Xeromyces bisporus stages of the A glaucus group are found in the genus Eurotium aw 0.75 0.61 0.61 0.92 0.91 0.90 ca 0.9 ca 0.90 ca 0.87 0.86 0.84 0.81 0.72 0.70 0.70 0.64 0.62 0.61 Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 47 (“dry-loving”) molds and osmophilic (preferring high osmotic pressures) yeasts have been reported to grow at aw values of 0.65 and 0.61, respectively (Table 3–5) When salt is employed to control aw , an extremely high level is necessary to achieve aw values below 0.80 (see Table 3–4) Certain relationships have been shown to exist among aw , temperature, and nutrition First, at any temperature, the ability of microorganisms to grow is reduced as the aw is lowered Second, the range of aw over which growth occurs is greatest at the optimum temperature for growth; and third, the presence of nutrients increases the range of aw over which the organisms can survive.32 The specific values given in Table 3–5, then, should be taken only as reference points, as a change in temperature or nutrient content might permit growth at lower values of aw Effects of Low aw The general effect of lowering aw below optimum is to increase the length of the lag phase of growth and to decrease the growth rate and size of final population This effect may be expected to result from adverse influences of lowered water on all metabolic activities because all chemical reactions of cells require an aqueous environment It must be kept in mind, however, that aw is influenced by other environmental parameters such as pH, temperature of growth, and Eh In their study of the effect of aw on the growth of Enterobacter aerogenes in culture media, Wodzinski and Frazier54 found that the lag phase and generation time were progressively lengthened until no growth occurred with a lowering of aw The minimum aw was raised, however, when the incubation temperature was decreased When both the pH and temperature of incubation were made unfavorable, the minimum aw for growth was higher The interaction of aw , pH, and temperature on the growth of molds on jam was shown by Horner and Anagnostopoulos.24 The interaction between aw and temperature was the most significant In general, the strategy employed by microorganisms as protection against osmotic stress is the intracellular accumulation of compatible solutes Halophiles (e.g., Halobacterium spp.) maintain osmotic equilibrium by maintaining the concentration of KCl in their cytoplasm equal to that of the suspending menstruum, and this is referred to as the “salt in cytoplasm” response Nonhalophiles accumulate compatible solutes (osmolytes) in a biphasic manner The first response is to increase K+ (and endogenously synthesized glutamate), and the second is to increase, either by de novo synthesis or by uptake, compatible solutes The latter are very soluble molecules that have no net charge at physiological pH, and they not adhere to or react with intracellular macromolecules (see reference 49) The three most common compatible solutes in most bacteria are carnitine, glycine betaine, and proline Carnitine may be synthesized de novo, but the other two are generally not Proline is synthesized by some Gram-positive bacteria while it is transported by Gram negatives The solubility of glycine betaine in 100 ml of water at 25◦ C is 160 g; it is 162 g for proline Glycine betaine is employed by more living organisms that the other two osmolytes noted The uptake of osmolytes is mediated by a transport system In L monocytogenes, glycine betaine is transporated by BetL (it couples betaine accumulation to a Na+ -motive) and Gbu (transports betaine) 48 Modern Food Microbiology whereas the transporter for carnitine is OpuC.1,49 Although some Gram-positive bacteria accumulate proline, it is concentrated to higher levels by Gram-negative bacteria The three transporter systems in E coli and S Typhimurium are PutP, ProP, and ProU, with ProP being the most effective It has been shown that the overproduction of proline by mutants of L monocytogenes did not lead to changes in mouse virulence.49 Under salt stress, L monocytogenes produces 12 proteins one of which is highly similar to the Ctc protein of B subtilis, and it is involved in osmotic stress tolerance in the absence of osmoprotectants in the medium.21 The sigma factor-B (δ B ; see Chapter 22) plays a major role in the regulation of carnitine utilization in L monocytogenes, but it is not essential for betaine utilization.20 Because it can grow at 4◦ C, evidence has been presented that low-temperature growth of L monocytogenes is aided by the accumulation of glycine betaine.29 The same is true for Yersinia enterocolitica, where osmotically stressed as well as cold-stressed cells accumulated osmolytes including glycine betaine.36 Temperature downshock and osmotic upshock caused a 30-fold uptake of radiolabeled glycine betaine.36 In at least one strain of L monocytogenes, glycine betaine transport is mediated by Gbu and BetL; and to a lesser extent OpuC.1 With regard to specific compounds used to lower water activity, results akin to those seen with adsorption and desorption systems (see Chapter 18) have been reported In a study on the minimum aw for the growth and germination of Clostridium perfringens, Kang et al.28 found the value to be between 0.97 and 0.95 in complex media when sucrose or NaCl was used to adjust aw but 0.93 or below when glycerol was used In another study, glycerol was found to be more inhibitory than NaCl to relatively salt-tolerant bacteria, but less inhibitory than NaCl to salt-sensitive species when compared at similar levels of aw in complex media.30 In their studies on the germination of Bacillus and Clostridium spores, Jakobsen and Murrell25 observed strong inhibition of spore germination when aw was controlled by NaCl or CaCl2 , but less inhibition when glucose or sorbitol was used, and very little inhibition when glycerol, ethylene, glycol, acetamide, or urea, were used The germination of clostridial spores was completely inhibited at aw = 0.95 with NaCl, but no inhibition occurred at the same aw when urea, glycerol, or glucose was employed In another study, the limiting aw for the formation of mature spores by B cereus strain T was shown to be about 0.95 for glucose, sorbitol, and NaCl, but about 0.91 for glycerol.26 Both yeasts and molds have been found to be more tolerant to glycerol than to sucrose.24 Using a glucose minimal medium and Pseudomonas fluorescens, Prior39 found that glycerol permitted growth at lower aw values than either sucrose or NaCl It was further shown by this researcher that the catabolism of glucose, sodium lactate, and dl-arginine was completely inhibited by aw values greater than the minimum for growth when aw was controlled with NaCl The control of aw with glycerol allowed catabolism to continue at aw values below that for growth on glucose In all cases where NaCl was used by this investigator to adjust the aw , substrate catabolism ceased at an aw greater than the minimum for growth, whereas glycerol permitted catabolism at lower aw values than the minimum for growth In spite of some reports to the contrary, it appears that glycerol is less inhibitory to respiring organisms than agents such as sucrose and NaCl Osmophilic yeasts accumulate polyhydric alcohols to a concentration commensurate with their extracellular aw According to Pitt,38 the xerophilic fungi accumulate compatible solutes or osmoregulators as a consequence of the need for high internal solutes if growth at a low aw is to be possible In a comparative study of xerotolerant and nonxerotolerant yeasts to water stress, Edgley and Brown19 found that Zygosaccharomyces rouxii responded to a low aw controlled by polyethylene glycol by retaining within the cells increasing levels of glycerol However, the amount did not change greatly, nor did the level of arabitol change appreciably by aw On the other hand, a nontolerant S cerevisiae responded to a lowering of aw by synthesizing more glycerol but retaining less The Z rouxii response to a low aw was at the level of glycerol permeation/transport, whereas that for S cerevisiae Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 49 was metabolic It appears from this study that a low aw forces S cerevisiae to divert a greater proportion of its metabolic activity to glycerol production accompanied by an increase in the amount of glucose consumed during growth In a later study, it was noted that up to 95% of the external osmotic pressure exerted on S cerevisiae, Z rouxii, and Debaryomyces hansenii may be counterbalanced by an increase in glycerol.43 Z rouxii accumulates more glycerol under stress, whereas ribitol remains constant It is known that the growth of at least some cells may occur in high numbers at reduced aw values, while certain extracellular products are not produced For example, reduced aw results in the cessation of enterotoxin B production by S aureus even though high numbers of cells are produced at the same time.50,51 In the case of Neurospora crassa, a low aw resulted in nonlethal alterations of permeability of the cell membrane, leading to a loss of several essential molecules.12 Similar results were observed with electrolytes or nonelectrolytes Overall, the effect of a lowered aw on the nutrition of microorganisms appears to be of a general nature where cell requirements that must be mediated through an aqueous milieu are progressively shut off In addition to the effect on nutrients, a lowered aw undoubtedly has adverse effects on the functioning of the cell membrane, which must be kept in a fluid state The drying of internal parts of cells would be expected to occur upon placing cells in a medium of lowered aw to a point where the equilibrium of water between cells and substrate occurs Although the mechanisms are not entirely clear, all microbial cells may require the same effective internal aw Those that can grow under extreme conditions of a low aw apparently so by virtue of their ability to concentrate salts, polyols, and amino acids (and possibly other types of compounds) to internal levels sufficient not only to prevent the cells from losing water, but that it may allow the cell to extract water from the water-depressed external environment For more information, see references 49, 51 Oxidation–Reduction Potential It has been known for decades that microorganisms display varying degrees of sensitivity to the oxidation–reduction potential (O/R, Eh) of their growth medium.23 The O/R potential of a substrate may be defined generally as the ease with which the substrate loses or gains electrons When an element or compound loses electrons, the substrate is oxidized, whereas a substrate that gains electrons becomes reduced: oxidation Cu ←− −→ Cu + e reduction Oxidation may also be achieved by the addition of oxygen, as illustrated in the following reaction: 2Cu + O2 → 2CuO Therefore, a substance that readily gives up electrons is a good reducing agent, and one that readily takes up electrons is a good oxidizing agent When electrons are transferred from one compound to another, a potential difference is created between the two compounds This difference may be measured by use of an appropriate instrument, and expressed as millivolts (mV) The more highly oxidized a substance, the more positive will be its electrical potential; the more highly reduced a substance, the more negative will be its electrical potential When the concentration of oxidant and reductant is equal, a zero electrical potential exists The O/R potential of a system is expressed by the symbol Eh Aerobic microorganisms require positive Eh values (oxidized) for growth, whereas anaerobes require negative 50 Modern Food Microbiology Figure 3–3 Schematic representation of oxidation–reduction potentials relative to the growth of certain microorganisms Eh values (reduced) (Figure 3–3) Among the substances in foods that help to maintain reducing conditions are –SH groups in meats and ascorbic acid, and reducing sugars in fruits and vegetables In regard to the maximum positive and negative mV values in Fig 3–3, not only are they not necessary for the growth of aerobes or anaerobes, but these extreme values can also be lethal to the respective group (see EO water section in Chapter 13) The O/R potential of a food is determined by the following: The characteristic O/R potential of the original food The poising capacity; that is, the resistance to change in potential of the food The oxygen tension of the atmosphere about the food The access that the atmosphere has to the food With respect to Eh requirements of microorganisms, some bacteria require reduced conditions for growth initiation (Eh of about −200 mV), whereas others require a positive Eh for growth In the former category are the anaerobic bacteria such as the genus Clostridium; in the latter belong aerobic ... substrate is oxidized, whereas a substrate that gains electrons becomes reduced: oxidation Cu ←− −→ Cu + e reduction Oxidation may also be achieved by the addition of oxygen, as illustrated in the following... require negative 50 Modern Food Microbiology Figure 3–3 Schematic representation of oxidation–reduction potentials relative to the growth of certain microorganisms Eh values (reduced) (Figure 3–3)... betaine is employed by more living organisms that the other two osmolytes noted The uptake of osmolytes is mediated by a transport system In L monocytogenes, glycine betaine is transporated by BetL